Use of glycosidase in preparation of a milk product

ABSTRACT

A method for making a milk product (e.g. a yogurt) comprising adding an effective amount of an N-linked glycosidase and/or an O-linked glycosidase to milk.

FIELD OF THE INVENTION

The present invention relates to a method for making a milk product(e.g. a yogurt) comprising adding an effective amount of an N-linkedglycosidase and/or a O-linked glycosidase to milk.

BACKGROUND ART

Milk products such as e.g. fermented milk products (e.g. yogurt) arewell known in the art.

As known in the art—properties such as viscosity and gel firmness areimportant properties for relevant milk products.

Generally speaking—if one can make more viscosity and/or gel firmness,using the same milk composition, one can make the same viscosity/gelfirmness using less milk (or other) solids, thus saving on raw material(or obtaining a higher yield).

For low fat fermented milk products such as e.g. low fat yogurt it maybe difficult to obtain an optimal preferred texture of the products—moreprecisely the problem may be to e.g. get a sufficient high textureviscosity in such low fat products.

Addition of protein, typically skim milk powder or whey based proteins,may be seen as a standard procedure to improve the texture in low fatyogurts. However, such a solution may be costly and does not fullycomply with the concept of a low fat/low calories product since theadded protein also contributes to the total energy content.

In relation to improvement of the texture—US2005/0095316A1 paragraph[0005] describes adding so-called texturing agents (thickeners, gellingagents) such as starch, pectin or gelatin. However, as known—to e.g.have extra pectin or gelatin in e.g. a yogurt product may not bepreferred.

Within the last years so-called texturizing cultures, based onextracellular polysaccharides (EPS) production, have markedly improvedthe viscosity of such low fat milk products—for instance WO2007/095958A1(Chr. Hansen A/S) describes that strains of Streptococcus thermophilussynthesize EPS that may give a desirable “ropy” or viscous texture tofermented milk products.

Accordingly, one may say that for e.g. low fat yogurt is the textureviscosity problem today as such pretty well solved by use of e.g. theseEPC producing cultures.

However, as described in the article of A. N. Hassan et al (J. DairySci: 86:1632-1638; 2003) the so-called gel firmness may decreasesignificantly when using such EPS producing cultures as compared to nonEPS producing cultures.

Accordingly—one may say that the situation today is that for e.g. lowfat yogurt products have the earlier viscosity problem essentially beensolved by the use of the EPS producing cultures—however, the use of theEPS cultures may have “created” a new problem in relation to thedecreased gel firmness.

In the article of A. N. Hassan et al is in the abstract referred tolower viscoelastic moduli—as known to the skilled person thisviscoelastic moduli feature relates to gel firmness, in the sense thatif the product has lower viscoelastic moduli it will have lower gelfirmness.

In FIG. 1 of the article is shown that that shear stress of the EPSyogurts was much higher, which to a person skilled in the art directlytranslates to a higher viscosity.

As discussed above—for milk products in general such as e.g. fermentedmilk products (e.g. yogurt)—the so-called gel firmness is a veryrelevant property.

For instance, low gel firmness may give an undesirable mouth feel to themilk product.

Further, if e.g. a yogurt has a low gel firmness is will appear thin andflow too readily—e.g. on the spoon or e.g. in a bowl.

Further, if e.g. a yogurt has a low gel firmness it may be prone tosyneresis (see below) due to whey separation.

As is well known in the art, milk clotting enzymes, such as the proteasechymosin (alternatively named rennin) is used to make cheese, wherechymosin causes coagulation and curd formation.

As is well known cheese making comprises three steps, or stages, thatall come about as the result of addition of a milk clotting enzyme: 1)the formation of a clot (or soft gel), the solidification, or firming ofthis clot, and the eventual expulsion of whey, the latter process alsocalled syneresis.

As evident—when making e.g. a yogurt one is generally not interested ingetting this syneresis effect—i.e. the separation of the milk into solidcurds and liquid whey.

Accordingly, use of a milk clotting enzyme like chymosin is generallynot preferred when one wants to make e.g. a yogurt.

US2005/0095317A1 (Danone) describes the use of a protease withkappa-caseinolytic activity in the production fermented milk productssuch as yogurt.

The protease may e.g. be chymosin—see e.g. [0028] of the US patentapplication.

The proteases (e.g. chymosin) hydrolyse casein in the milk—accordinglyone should prima facie have believed that the proteases (e.g. chymosin)could have given rise to a for yogurt production unwanted syneresiseffect.

However, section [0008] explains that surprisingly and unexpectedly itwas shown that use of proteases such as e.g. chymosin “improve thetexture, and in particular to increase the viscosity of yogurts andfermented milks, without as a result inducing syneresis which would beunacceptable for such fermented dairy products”.

US2005/0095316A1 (Danone) essentially relates to the same technicalteaching as in US2005/0095317A1 (Danone) discussed above—however, inthis US application the relevant proteases are defined as bacterialproteases (chymosin is from cows—i.e. it is not a bacterial protease).

U.S. Pat. No. 7,560,127B2 (DSM) describes use of special deglycosylationenzymes in cheese production. The deglycosylation enzymes are defined asenzymes that can deglycosylate the kappa casein (κ-casein) present inthe milk. As discussed below—casein is a protein with so-called O-linkedglycosylation. Accordingly, the deglycosylation enzymes mentioned inthis US patent are enzymes that can deglycosylate O-linked glycosylatedproteins such as kappa-casein (kappa-casein is a so-called O-linkedglycoprotein).

The US patent reads on column 1, lines 51-58:

“It was surprisingly found that a deglycosylation of κ-casein will leadto clotting as it are the sugars associated with κ-casein that carry thenegative charge which stabilize the casein micelles. Clotting of themilk in this way results in a process in which a larger part of theκ-casein is retained in the cheese and a higher yield can be obtainedthan using proteolytic activity of chymosin.”

In short, one may say that this U.S. Pat. No. 7,560,127B2 patentessentially describes that one may get the, for cheese production,required clotting by using the mentioned O-linked relateddeglycosylation enzymes instead of chymosin. Since this U.S. Pat. No.7,560,127B2 patent relates to production of cheese and in the claim 1 itis said that one may get the cheese without using a protease (such aschymosin) the skilled person will implicitly understand that the use ofthe O-linked related deglycosylation enzymes must lead to a substantialamount of syneresis.

It is here relevant to note that U.S. Pat. No. 7,560,127B2 relates toproduction of cheese, where a substantial amount of syneresis isrequired. Consequently, a skilled person would not expect it to apply tofermented milk product like yogurt, where syneresis is generally highlyundesirable.

Further, U.S. Pat. No. 7,560,127B2 does not explicitly say anythingabout the herein relevant gel firmness property—i.e. one may say it onlyexplicitly relates to the clotting/syneresis effect attributed to use ofthe described O-linked deglycosylation enzymes instead of chymosin.

The article of E. Cases et al (Journal of Food Science; Vol. 68, Nr. 8,2003, Pages 2406-2410) describes deglycosylation of chemically acidifiedmilk with an O-linked deglycosylation enzyme (neuraminidase; EC3.2.1.18).

The milk is chemically acidified with the chemical GDL (see p. 2407,section “Acid milk coagulation”)—accordingly, there is in the E. Caseset al article not described a “fermented milk” product inoculated withrelevant microorganisms (e.g. a yogurt).

The article of E. Cases et al describes that the use of the O-linkeddeglycosylation enzyme neuraminidase (EC 3.2.1.18) gave higher final gelfirmness to the enzymatically treated and chemically acidified milk.

It is here relevant to note that the article of E. Cases et al does notsay anything of herein relevance with respect to the possible syneresiseffect of using the O-linked deglycosylation enzyme neuraminidase (EC3.2.1.18).

In the E. Cases et al article is not provided relevant evidence for thepurity of the used neuraminidase (EC 3.2.1.18) O-linked deglycosylationenzyme preparation.

In view of this—it is submitted, that the enzyme preparation used mayhave comprised some relevant protease enzyme activity and that thisprotease enzyme activity could have been responsible for the describedgel firmness effect—as discussed above, US2005/0095317A1 (Danone)describes that the use of a protease may improve the gel firmness.

The relevance of this suspicion is further underlined by that fact thatClostridium perfringens, the microorganism that the neuraminidase in theE. Cases et al. article was derived from (see Materials and Methodsection) is known to contain more than 140 proteolytic enzymes—see e.g.the authoritative Merops peptidase database(http://merops.sanger.ac.uk/cgi-bin/speccards?sp=sp000283;type=peptidase).

With no information on purity of the enzyme preparation provided, itthus seems very plausible that it was not purified to the exclusion ofall relevant proteolytic enzymes that could in themselves have had theeffect on gel firmness described in the E. Cases et al article.

As known in the art—the O-linked deglycosylation enzyme neuraminidase(EC 3.2.1.18) may also have some N-linked glycosidase activity. However,the E. Cases et al article discussed above only refers to the O-linkedglycosidase activity of neuraminidase (EC 3.2.1.18) when herein relevantenzymatic activities of neuraminidase is discussed in he E. Cases et alarticle.

EP1489135A1 may be seen as relating to use of deglycosylated oleuropein(obtained from Olive leaf extracts) for increasing gel firmness ofacidified whey milk and yogurt. Beta-glycosidase (beta-1,6-glucosidase)and lactase are in this document “simply” used to deglycosylate theoleuropein—i.e. the enzymes are here not the active component forincreasing the gel firmness in the milk/yogurt. Oleuropein is not aprotein/peptide with enzymatic activity. Beta-glycosidase is neither anN- nor O-linked glycosidase. The method of the present invention doespreferably not comprise addition of an activated olive leaf extract (orother extracts) as disclosed in EP1489135A1 to an animal milk substrate.

SUMMARY OF THE INVENTION

The problem to be solved by the present invention may be seen in theprovision of a new method to improve/increase the gel firmness of arelevant milk product—e.g. a fermented milk product like yogurt—inparticular a low fat fermented milk product like a low fat yogurt.Further—the new method should preferably not negatively affect theviscosity of the product.

The solution is based on that the present inventors have identified thatby use of a deglycosylation enzyme (i.e. a glycosidase) one can get amilk product (e.g. a yogurt) with significant increased/improved gelfirmness. Further, the present inventors identified that use of hereinrelevant glycosidases may be done in a manner, wherein there is nosignificant syneresis effect—as discussed above, to have no significantsyneresis effect is a big advantage e.g. in relation to making afermented milk product such as an yogurt. In particular, in relation touse of O-linked glycosidase it was a surprise to the inventors that itcould be used without a significant syneresis effect. The presentinventors also identified that use of the deglycosylation enzyme doesnot negatively affect the viscosity of the product.

In working Example 4 herein is shown that use of the glycosidasePNGase-F gave a low fat yogurt with significant increased/improved gelfirmness without any negative effect on the viscosity of the low fatyogurt.

As described in working Example 1 herein—the glycosidase enzymepreparations used in the working Examples herein were analyzed and foundfree of contaminants—i.e. they did not comprise contaminants such asprotease enzyme activity.

It is here interesting to note that PNGase-F is a glycosidase that workson “N-linked glycans”—i.e. deglycosylate N-linked glycoproteins.

As discussed above—casein is a glycoprotein comprising O-linked glycansand PNGase-F can therefore as such not deglycosylate casein—accordingly,the reason for that PNGase-F gives the herein positive gel firmnessresult cannot as such be due to a mechanism relating to deglycosylationof casein as e.g. discussed in U.S. Pat. No. 7,560,127B2 (see above).

In summary and without being limited to theory—the fact that the presentinventors demonstrated that a N-linked glycosidase like PNGase-F worksin the present context (i.e. gives improved gel firmness) illustratesthat the basic mechanism behind getting the herein relevant improved gelfirmness is completely different from the “deglycosylation of casein”based mechanism suggested in U.S. Pat. No. 7,560,127B2 with respect touse O-linked glycosidases as a substitute for chymosin in thepreparation of cheese (see above).

Said in other words, one may say that the fact that a N-linkedglycosidase like PNGase-F works in the present context (i.e. givesimproved gel firmness) may be seen as highly surprising for a skilledperson in view of the prior art such as e.g. U.S. Pat. No. 7,560,127B2.

Without being limited to theory—a possible explanation for that use of aglycosidase as such can give the herein relevant improved gel firmnessis that the glycosidase removes glycans from relevant not casein wheyproteins.

For instance, an N-linked glycosidase like PNGase-F may remove N-glycansfrom some of the whey glycoproteins such as α-lactalbumin which areknown to be N-glycosylated. The whey proteins also to some extend engagein the protein-network, that gives rise to the property gel firmness.

Removal of hydrophilic glycans and charge changes imposed by thedeamination reaction may favor the formation of a more rigid proteinnetwork and thereby one gets improved gel firmness.

Without being limited to theory—it is believed that some herein relevantwhey glycoproteins also comprise O-linked glycans.

Accordingly, the present inventors tested if an O-linked glycosidasealso could give the herein relevant improved gel firmness.

In working Example 5 herein is demonstrated that also use of an O-linkedglycosidase like GalNAC can give the herein relevant improved gelfirmness.

From a theoretically point of view—one would believe that a hereinrelevant advantage of using an N-linked glycosidase like PNGase-F wouldbe a limited (or no) unwanted syneresis effect e.g. in relation tomaking a yogurt product.

One reason for this theoretically point of view is that a N-linkedglycosidase like PNGase-F does not work on casein and as discussed ine.g. U.S. Pat. No. 7,560,127B2 one may get the, for cheese production,required clotting (syneresis) by using the mentioned O-linked relateddeglycosylation enzymes (to deglycosylate casein) instead of chymosin.

In working Example 4 herein was demonstrated that the N-linkedglycosidase PNGase-F gave no herein unwanted syneresis effect inrelation to making a yogurt.

The present inventors tested the herein unwanted syneresis effect inrelation to e.g. making a yogurt for an O-linked glycosidase likeGalNAC.

As shown in working Example 6 herein—one may say to the surprise of thepresent inventors—the there was no herein unwanted syneresis effect inrelation to making a yogurt when the 0-linked glycosidase GalNAC wasused for yogurt production.

In working Example 7 herein—is demonstrated that use of a glycosidase(here PNGase-F) gave the herein relevant improved gel firmness in bothlactic acid bacteria (LAB) and chemical acidified milk.

Without being limited by theory, this suggests that the effect of theglycosidase on gel firmness is a physical effect (i.e. deglycosylationof whey proteins as discussed above)—rather than a biological effect(i.e. an effect, wherein the glycosidase essentially only affectssomething in relation to the functionality of the LAB as such).

In the working Examples discussed above were used already heat treatedmilk—i.e. the glycosidase enzyme was added to the milk after therelevant heat treatment.

Without being limited to theory—a glycosidase like e.g. PNGase-F shouldalso work (i.e. give the herein relevant gel firmness improvement) whenadded to raw milk (i.e. not heat treated milk) followed by a hereinrelevant heat treatment.

Accordingly, an first aspect of the present invention relates to amethod for making a milk product comprising the following step:

(i): adding a N-linked glycosidase and/or a O-linked glycosidase to amilk substrate; and(ii) optionally acidifying (e.g. fermenting) the milk substrate, and/oroptionally performing adequate step(s) to get a milk product, whereinthe adequate step(s) is/are performed under conditions, wherein theeffective amount of the glycosidase gives—as a result of itspresence—increased gel firmness to the dairy milk product.

The milk substrate may be selected for the group consisting of milk fromanimals (such as cows, sheep, ewes, goats, buffaloes or camels) and milkof plant origin (such as soy milk, oak milk, rice milk, almond milk).

An embodiment of the first aspect relates to a method for making a dairyanimal milk product comprising following steps:

-   -   (i): adding an effective amount of a N-linked glycosidase and/or        a O-linked glycosidase to an animal milk substrate; and    -   (ii): performing adequate step(s) to get a dairy animal milk        product, wherein the adequate step(s) is/are performed under        conditions, wherein the effective amount of the glycosidase        gives—as a result of its presence—increased gel firmness to the        dairy milk product;        with the proviso that if the glycosidase is only an O-linked        glycosidase (such as α-galactosidase, N-acetyl-galactosaminidase        [GalNAC] and neuraminidase) capable of performing        deglycosylation of κ-casein present in the milk then is the        dairy animal milk product a fermented milk product inoculated        with relevant microorganisms (a fermented milk product is not a        cheese product with a significant elimination of milk serum).

The term “the effective amount of the glycosidase gives—as a result ofits presence—increased gel firmness to the dairy milk product” of step(ii) of the first aspect may alternatively be termed “the effectiveamount of the glycosidase gives—as a result of its presence—improved gelfirmness to the dairy milk product”.

The term “performing adequate step(s) to get a milk product” of step(ii) shall be understood as relevant adequate step(s) to make a milkproduct of interest (e.g. a yogurt).

It is evident that the skilled person is perfectly aware of suchadequate step(s)—that for instance in relation to making a yogurtcomprises inoculation with suitable lactic acid bacteria (LAB) cultures.

It is routine work for the skilled person to measure gel firmness of amilk product of interest (e.g. a yogurt).

In working Example 2 herein is provided a suitable standard method formeasurement of gel firmness of a milk product of interest—preferably theherein relevant gel firmness is measured according to the method of thisExample 2.

The article of A. N. Hassan et al (3. Dairy Sci: 86:1632-1638; 2003)discussed above also describes a suitable standard method formeasurement of gel firmness.

See e.g. the materials and method section of the article, where elasticmodulus and viscoelastic modulus are determined—as can be seen inExample 2 herein, the parameters elastic modulus and viscoelasticmodulus are used to determine gel firmness and from which one canoptionally obtain the so-called complex modulus, as described in Example2 herein.

Since it is easy for the skilled person to measure gel firmness of amilk product of interest—it is of course also easy for the skilledperson to determine the requirement of step (ii) of the method of thefirst aspect relating to if:

“the effective amount of the glycosidase gives—as a result of itspresence—improved gel firmness to the milk product”.

In order to determine this requirement—the skilled person shall simplyperform the “adequate step(s) to get a milk product” of step (ii) withand without presence of the effective amount of the glycosidase—and thendetermine the herein relevant gel firmness effect of the presence of theadded amount of glycosidase.

If there is improved/increased gel firmness then there have been addedan effective amount of the glycosidase to the milk substrate inaccordance with step (i) of the first aspect and the adequate step(s) ofstep (ii) have also been performed under conditions, wherein theeffective amount of the glycosidase gives—as a result of itspresence—improved gel firmness to the dairy milk product.

As known to the skilled person—different methods to measure gel firmnessmay give different results in absolute values. However, in relation tomeasurement of improved gel firmness as discussed herein one isessentially measuring a relative improvement of the gel firmness—i.e.the improvement with and without presence of the effective amount of theglycosidase.

As understood by the skilled person—the method to measure gel firmnessas e.g. described in working Example 2 herein and the article of A. N.Hassan et al will (within relatively minor measurement uncertainties)give the same relative results—i.e. independently of the specificmeasurement method used one will get the same result with respect to therelative improvement of the gel firmness.

The proviso of the method of the first aspect may be seen as adisclaimer in relation to above discussed U.S. Pat. No. 7,560,127B2 andabove discussed article of E. Cases et al (Journal of Food Science; Vol.68, Nr. 8, 2003, Pages 2406-2410).

As discussed above—there is in the E. Cases et al article not describeda “fermented milk” product inoculated with relevant microorganisms (e.g.a yogurt).

As discussed above—U.S. Pat. No. 7,560,127B2 does not explicitly sayanything about the herein relevant gel firmness property—i.e. one maysay it only relates to the clotting/syneresis effect attributed to useof the described O-linked deglycosylation enzymes instead of chymosin.

However, one may say that by adding the in U.S. Pat. No. 7,560,127B2described O-linked deglycosylation enzymes to a milk substrate duringthe process for making the cheese milk product—there could theoreticallyimplicitly have been added an effective amount of glycosidase theremaybe could have given a herein relevant improved gel firmness.

The proviso relates to the situation, wherein the glycosidase is only anO-linked glycosidase—U.S. Pat. No. 7,560,127B2 does not describeanything of herein relevance in relation to use of N-linkedglycosidase—accordingly, if the effective amount of a glycosidase (i.e.covering one or more glycosidase) added in step (i) of first aspectwould be e.g. a mixture of a N-linked glycosidase and a O-linkedglycosidase then it would not be a situation, wherein the glycosidase isonly an 0-linked glycosidase.

In the present context—the skilled person can routinely identify if amilk product is a cheese product as described in U.S. Pat. No.7,560,127B2 or another herein relevant milk product such as e.g. afermented milk product like e.g. a yogurt.

DEFINITIONS

The term “glycan” refers to a polysaccharide or oligosaccharide. Glycanscan be homo or heteropolymers of monosaccharide residues, and can belinear or branched. Glycan may also be used to refer to the carbohydrateportion of a glycoconjugate, such as a glycoprotein, glycolipid, or aproteoglycan.

The term “glycoproteins” are proteins that contain oligosaccharidechains (glycans) covalently attached to polypeptide side-chains. Thecarbohydrate is attached to the protein in a cotranslational orposttranslational modification.

The term “glycosidase” (also called glycoside hydrolase) refers to anenzyme that catalyzes the hydrolysis of the glycosidic linkage/bond—aglycosidic bond is a type of covalent bond that joins a carbohydrate(sugar) molecule to another group, which may or may not be anothercarbohydrate. A glycosidase that partially or completely deglycosylateN-linked glycans may herein be termed an N-linked glycosidase.Similarly, a glycosidase that partially or completely deglycosylateO-linked glycans may herein be termed an O-linked glycosidase.

The term N-linked glycosidase is a well defined term in the art and theskilled person knows if a specific glycosidase of interests is aN-linked glycosidase or not.

Similarly, the term O-linked glycosidase is a well defined term in theart and the skilled person knows if a specific glycosidase of interestsis an O-linked glycosidase or not.

A glycosidase may herein also be termed a deglycosylation enzyme.

The term “glycosylation” is the enzymatic process that attaches glycansto proteins, lipids, or other organic molecules.

The term “N-linked glycans” refers to glycans attached to a nitrogen ofnormally asparagine or arginine side chains.

The term “O-linked glycans” refers to glycans attached to the hydroxyoxygen of normally serine, threonine, tyrosine, hydroxylysine, orhydroxyproline side chains, or to oxygens on lipids such as ceramide.

The term “lactic acid bacterium” designates a gram-positive,microaerophilic or anaerobic bacterium, which ferments sugars with theproduction of acids including lactic acid as the predominantly producedacid, acetic acid and propionic acid. The industrially most usefullactic acid bacteria are found within the order “Lactobacillales” whichincludes Lactococcus spp., Streptococcus spp., Lactobacillus spp.,Leuconostoc spp., Pseudoleuconostoc spp., Pediococcus spp.,Brevibacterium spp., Enterococcus spp. and Propionibacterium spp.Additionally, lactic acid producing bacteria belonging to the group ofthe strict anaerobic bacteria, bifidobacteria, i.e. Bifidobacteriumspp., are generally included in the group of lactic acid bacteria. Theseare frequently used as food cultures alone or in combination with otherlactic acid bacteria,

The term “milk substrate” may be any raw and/or processed milk materialthat can be subjected to enzymatic treatment (and possibly fermentation)according to the method of the invention. Thus, useful milk substratesinclude, but are not limited to, solutions/suspensions of any milk ormilk like products comprising protein, such as whole or low fat milk,skim milk, buttermilk, reconstituted milk powder, condensed milk, driedmilk, whey, whey permeate, lactose, mother liquid from crystallizationof lactose, whey protein concentrate, or cream. Obviously, the milksubstrate may originate from any animal (mammal) or non-animal source,e.g. being substantially pure milk, or reconstituted milk powder.Preferably, at least part of the protein in the milk substrate isproteins naturally occurring in milk, such as casein or whey protein.However, part of the protein may be proteins which are not naturallyoccurring in milk. Prior to fermentation, the milk substrate may behomogenized and pasteurized according to methods known in the art.

The term “milk” is to be understood as the lacteal secretion obtained bymilking any mammal, such as cows, sheep, ewes, goats, buffaloes orcamels. In a preferred embodiment, the milk is cow's milk. The term milkalso comprises “milks” of non-animal or plant origin (eg from avegetable or cereal source), such as soy milk, oak milk, rice milk,almond milk. Other sources are cotton, wheat, malt, corn, potato, bean,lupin, and sorghum. Optionally the milk is acidified, e.g. by additionof an acid (such as citric, acetic or lactic acid), or mixed, e.g. withwater. The milk may be raw or processed, e.g. by filtering, sterilizing,pasteurizing, homogenizing etc, or it may be reconstituted dried milk.An important example of “bovine milk” according to the present inventionis pasteurized cow's milk. It is understood that the milk may beacidified, treated with glycosidase, mixed or processed before, duringand/or after the inoculation with bacteria.

In the present context, the term dairy product is a product made bytreatment of a milk substrate with an N or O linked glucosidase,optionally the product is also fermented or acidified. The term includesfermented milk products (which can be drinkable, stirred or set) andcheeses.

“Homogenizing” as used herein means intensive mixing to obtain a solublesuspension or emulsion. If homogenization is performed prior tofermentation, it may be performed so as to break up the milk fat intosmaller sizes so that it no longer separates from the milk. This may beaccomplished by forcing the milk at high pressure through smallorifices.

“Fermentation” in the methods of the present invention means theconversion of carbohydrates into alcohols or acids through the action ofa microorganism. Preferably, fermentation in the methods of theinvention comprises conversion of lactose to lactic acid.

Lactic acid bacteria, including bacteria of the species Lactobacillussp. and Streptococcus thermophilus, are normally supplied to the dairyindustry either as frozen or freeze-dried cultures for bulk starterpropagation or as so-called “Direct Vat Set” (DVS) cultures, intendedfor direct inoculation into a fermentation vessel or vat for theproduction of a dairy product, such as a fermented milk product. Suchcultures are in general referred to as “starter cultures” or “starters”.

Optionally, the fermented milk substrate may be subjected to heattreatment to inactivate the microorganism.

Fermentation processes to be used in production of fermented milkproducts are well known and the person of skill in the art will know howto select suitable process conditions, such as temperature, oxygen,amount and characteristics of microorganism(s) and process time.Obviously, fermentation conditions are selected so as to support theachievement of the present invention, i.e. to obtain a fermented milkproduct.

In the present context, a yoghurt starter culture is preferably abacterial culture which comprises at least one Lactobacillus strain,e.g. a Lactobacillus bulgaricus strain, and at least one Streptococcusthermophilus strain. In accordance herewith, a yogurt is a fermentedmilk product obtainable by inoculating and fermenting milk with aLactobacillus strain and a Streptococcus thermophilus strain.

The term “spoonable” should be understood as to be consumed using aspoon. The term “spoonable milk product” includes stirred products. Theterm “stirred type product” specifically refers to a fermented milkproduct which sustains a mechanical treatment after fermentation,resulting in a destructuration and liquefaction of the coagulum formedunder the fermentation stage. The mechanical treatment is typically butnot exclusively obtained by stirring, pumping, filtrating orhomogenizing the gel, or by mixing it with other ingredients.

The term “set-type product” includes a product based on milk which hasbeen inoculated with a starter culture, e.g. a starter culture, andpackaged next to the inoculating step and then fermented in the package.

The term “drinkable product” includes beverages such as “drinkingyoghurt” and similar. The term “drinking yoghurt” typically covers amilk product produced by fermentation by the combination ofLactobacillus species and Streptococcus thermophilus. Drinking yoghurttypically has a milk solid non-fat content of 8% or more. Furthermore,the live culture count for drinking yoghurt drinks is typically at least10E6 cell forming units (CFU) pr ml.

DETAILED DESCRIPTION OF THE INVENTION Dairy Animal Milk Product

Generally speaking—the pH of a herein preferred milk product, includinga dairy animal milk product (i.e. a product based on animal milk), is apH from pH 3 to pH 6.5—more preferably from pH 3.5 to pH 5.75.

As know to the skilled person—one may get the relevant pH of a milkproduct by e.g. fermenting with a suitable lactic acid bacteria culture.

However, as known to the skilled person one may simply add a suitableacid (such as lactic acid) to get the required pH.

Alternative one may add a lactone (e.g. GDL lactone) to get the requiredpH or use other suitable known methods (e.g. enzymatic methods orpressureatiation with carbon dioxide) to get the required pH.

As discussed above—the use of a glycosidase to improve gel firmness asdescribed herein may be particular useful in relation to so-called lowfat milk products.

Accordingly, in a preferred embodiment the milk substrate used in step(i) of the method of the first aspect is a milk substrate with a low fatcontent—i.e. with a fat content of less than 3.5% fat, more preferablywith a fat content of less than 1.5% and even more preferably with a fatcontent of less than 0.75%.

As discussed above—an advantage of the use of a glycosidase to improvegel firmness as described may be that one does not need to increase theprotein content of e.g. a low fat milk product in order to getsufficient adequate gel firmness and thereby further minimizing thetotal calorific/energy content of the final low fat milk product (e.g. alow fat yogurt).

Accordingly, in a preferred embodiment the final milk product (e.g.yogurt) has a total calorific/energy content of less than 150 kilocalories per 100 g of milk product, more preferably the final milkproduct (e.g. yogurt) has a total calorific/energy content of less than100 kilo calories per 100 g of the milk product.

As known—it is routine work for the skilled person to determine thecalories content of a milk product of interest.

A suitable example of a milk product is a fermented milk product or acheese.

In a preferred embodiment, the milk product is a fermented milk product.

In a preferred embodiment—the fermented milk product is at least onefermented milk product selected from the group consisting of: yogurt,alternate culture yogurt, butter milk, acidophilus milk, kefir, kumysand quark. Most preferably, fermented milk product is a yogurt.

In the present context—the terms “yogurt” and “fermented milk” havetheir usual meanings. In US2005/0095316A1 and US2005/0095317A1 (bothDanone) are these terms defined in accordance with a relevant officialdecree/regulation in France—below is essentially referred to the samestandard known definitions of these terms.

As known to the skilled person—to obtain a “yogurt or fermented milk”product it is in particular recalled that there must not be asignificant elimination of milk serum and that there must be a heattreatment at least equivalent to pasteurization.

A suitable relevant heat treatment for making a fermented milk productsuch as e.g. a yogurt is, for example, a heat treatment of from 85 to98° C. for 15 seconds to 30 minutes.

Because of the application of a heat treatment which is at leastequivalent to standard pasteurization, milk serum proteins of the milksubstrate are denaturated more or less (from 25 to 99% of them,approximately).

As evident to a skilled person—since there for a yogurt or fermentedmilk product “must not be a significant elimination of milk serum”—ayogurt or fermented milk product is not a cheese product as described inU.S. Pat. No. 7,560,127B2 (see above).

The term “fermented milk” relates to dairy product prepared with skimmedor unskimmed milks or skimmed or unskimmed, concentrated or powderedmilks, enriched or not enriched with milk constituents, which has beensubjected to heat treatment at least equivalent to pasteurization,inoculated with microorganisms belonging to the species that is or arecharacteristic of each product.

The amount of free lactic acid which they contain should preferably notbe less than 0.6 gram per 100 grams at the time of sale to the consumer.

Fermented milks should preferably be kept, up to the time of sale to theconsumer, at a temperature capable of preventing them spoiling.

The term “yogurt” denotes fermented milk obtained, according to fair andtraditional practices, preferably by the development of specificthermophilic lactic acid bacteria only, such as e.g. Lactobacillusdelbrueckii subsp. bulgaricus and Streptococcus thermophilus, whichpreferably should be inoculated simultaneously and preferably be live inthe finished product, at a rate of preferably at least 10 millionbacteria per gram expressed in relation to the milk-containing portion.

A fermented milk product is normally obtained by

(A): inoculating from 10⁵ to 10¹³ cfu/ml (preferably 10⁶ to 10¹¹ cfu/ml)of lactic acid bacteria (LAB) culture to the animal milk substrate; and(B): fermenting the milk substrate from 2 to 120 hours at a temperaturefrom 10° C. to 55° C.

As known to the skilled person—suitable species of lactic acid bacteriainclude Bifidobacterium, Lactobacillus (such as Lactobacillusdelbrueckii subsp. bulgaricus, Lactobacillus acidophilus, Lactobacilluscasei or Lactobacillus helveticus), Streptococcus (such as Streptococcusthermophilus), Lactococcus (such as Lactococcus lactis), Leuconostoc(such as Leuconostoc lactis, Leuconostoc mesenteroides).

When the milk substrate is inoculated with a ferment made up of strainsof Lactobacillus delbrueckii subsp. bulgaricus and of Streptococcusthermophilus, the product is generally understood to be a yogurt.

As known in the art—the term “alternate culture yogurt” refers to afermented milk product made by using cultures of Streptococcusthermophilus and any Lactobacillus species.

As known in the art—the term “acidophilus milk” refers to a fermentedmilk product made by using culture of Lactobacillus acidophilus.

As known in the art—the term “Kefir” refers to a fermented milk productmade by using starter culture prepared from kefir grains, Lactobacilluskefiri, species of the genera Leuconostoc, Lactococcus and Acetobactergrowing in a strong specific relationship. Kefir grains constitute bothlactose fermenting yeasts (Kuyveromyces marxianus) andnon-lactose-fermenting yeasts (Saccharomyces unisporus, Saccharomycescerevisiae and Saccharomyces exiguus).

As known in the art—the term “Kumys” refers to a fermented milk productmade by using cultures of Lactobacillus delbrueckii subsp. bulgaricusand Kluyveromyces marxianus.

In a preferred embodiment—the milk product is a yogurt, wherein theyogurt is made by inoculation with a yogurt lactic acid bacteria culturethat comprises Lactobacillus delbrueckii subsp. bulgaricus andStreptococcus thermophilus capable of synthesizing extracellularpolysaccharides (EPS)—this preferred embodiment may be particularrelevant if the yogurt is a low fat yogurt, i.e. wherein the milksubstrate used in step (i) of the method of the first aspect is a milksubstrate with a low fat content—i.e. with a fat content of less than3.5% fat, more preferably with a fat content of less than 1.5% and evenmore preferably with a fat content of less than 0.75%.

As discussed above—the prior art describes a number of such strains ofStreptococcus thermophilus that produce EPS—see e.g. WO2007/095958A1(Chr. Hansen A/S).

Accordingly, the skilled person can routinely identify a number of suchEPS producing strains and he can also routine identify if a specificstrain of interest is capable of synthesizing EPS or not.

An example of a herein possible relevant theoretical business scenariocould be that a company makes a milk concentrate/powder by use of aglycosidase as described herein and then sells this milkconcentrate/powder to e.g. a yogurt producer that use this in theiryogurt production to get a yogurt with improved/increased gelfirmness—i.e. they may get the improved gel firmness without any extraaddition as such of glycosidase during the yogurt production as such.

As understood by the skilled person—such a theoretical business scenariowould be an example of a method within the scope of the method of thefirst aspect as discussed herein. The milk concentrate/powder may beseen as an example of a dairy animal milk product of the method of thefirst aspect. Further, as understood by the skilled person in thepresent context—the final yogurt will have the improved/increased gelfirmness due to the previous addition of the glycosidase to themilk—i.e. the yogurt producer will also perform actions within the scopeof the method of the first aspect as discussed herein.

Glycosidase

As discussed above—the term “glycosidase” (also called glycosidehydrolase) refers to an enzyme that catalyzes the hydrolysis of theglycosidic linkage/bond—a glycosidic bond is a type of covalent bondthat joins a carbohydrate (sugar) molecule to another group, which mayor may not be another carbohydrate.

As described above—a glycosidase may herein also be termed adeglycosylation enzyme.

The glycosidase may be a natural glycosidase or it may be avariant/mutated of a natural glycosidase—as known to the skilled person,one may make mutated variants of a enzyme of interest (here aglycosidase) to e.g. improve the stability of the enzyme whilemaintaining the key enzymatic activity (here glycosidase activity) ofthe enzyme.

In order to e.g. get a minimum of unwanted syneresis (in particular ifthe milk product is a fermented milk product such as a yogurt)—it may bepreferred that the glycosidase is an N-linked glycosidase.

As discussed above, the term N-linked glycosidase is a well defined termin the art and the skilled person knows if a specific glycosidase ofinterests is a N-linked glycosidase or not. Further the prior artdescribes a number of different herein suitable N-linked glycosidases.

Examples of a herein suitable N-linked glycosidase may be at least oneglycosidase selected from the group consisting of:Peptide-N(4)-(N-acetyl-beta-glucosaminyl)asparagine amidase (EC number:3.5.1.52; alternative names: N-Glycosidase-F or PNGase-F) andEndo-β-N-acetylglucosaminidase H (EC number: 3.2.1.96; alternative nameENDO-H).

The immediately above described N-linked glycosidases may in the presentcontext be described as glycosidases that have N-linked glycosidaseactivity and no herein significant O-linked glycosidase activity.

Accordingly, it may herein be preferred that the N-linked glycosidase isan N-linked glycosidase that have no herein relevant O-linkedglycosidase activity (such as no O-linked glycosidase activity).

The N-Glycosidase-F, also known as PNGase-F, used in the process, is anasparagine amidase (EC 3.5.1.52) that may be derived from Flavobacteriummesingosepticum. It catalyses the complete and intact cleavage ofN-linked oligosaccaharides from glycoproteins. It may be derived as acommercial product from New England Biolabs Inc. under the name PNGase-For produced recombinantly in a strain like Escherichia coli as we havedone using the plasmid and method described in working Example 1 herein.

Endo-β-N-acetylglucosaminidase H (EC 3.2.1.96), also known as ENDO-H,may be derived from Streptomyces plicatus. ENDO-H catalyses thehydrolysis of the glycosidic bond between the two N-acetylglycosaminesof N-linked glycosylations. It may be derived as a commercial productfrom New England Biolabs Inc. under the name ENDO-H.

Examples of a herein suitable O-linked glycosidase may be at least oneglycosidase selected from the group consisting of:α-N-acetyl-galactosaminidase (EC number: 3.2.1.49; alternative name:GalNAC); α-galactosidase (EC number: 3.2.1.22); and neuraminidase (ECnumber: 3.2.1.18).

GalNAC is a highly specific exoglycosidase that catalyzes the hydrolysisof α-linked D-N-acetyl-galactosamine residues from. It may be derived asa commercial product from New England Biolabs Inc.

As discussed above, the term O-linked glycosidase is a well defined termin the art and the skilled person knows if a specific glycosidase ofinterests is a O-linked glycosidase or not. Further the prior artdescribes a number of different herein suitable O-linked glycosidases.

The effective amount/activity of a glycosidase is herein determinedaccording to the art.

According to the art—for a N-linked glycosidase (such as e.g. PNGase-Fand Endo-H) one activity unit is defined as the amount of enzymerequired to remove >95% of the carbohydrate from 10 μg of denaturedRNase-B in 1 hour at 37° C. in a total reaction volume of 10 μl.

For GalNAC (an O-linked glycosidase) one activity unit is defined as theamount of enzyme required to cleave >95% of the terminalα-D-N-acetyl-galactosamine from 1 nmol(GalNAcα1-3)(Fucα1-2)Galα1-4Glc-7-amino-4-methyl-coumarin (AMC), in 1hour at 37° C. in a total reaction volume of 10 μl.

A number of herein relevant glycosidase enzymes are commerciallyavailable from the company New England Biolabs—reference is also made tothe product catalogue New England Biolabs (as e.g. available on-line ontheir web-page) for further details in relation to specific standarddefinitions of herein relevant glycosidase activity units.

Step (i) of the Method of First Aspect

Step (i) of the herein described method of the first aspect reads: “(i):adding an effective amount of an N-linked glycosidase and/or an O-linkedglycosidase to an animal milk substrate”.

As understood by the skilled person in the present context—the N-linkedglycosidase and/or an O-linked glycosidase is normally added to theanimal milk substrate as a substantial pure glycosidase composition—e.g.a glycosidase composition, wherein the glycosidase activity representsat least 5% of the total enzymatic activity of the glycosidasecomposition as such.

It may be a glycosidase composition, wherein the glycosidase activityrepresents at least 25% of the total enzymatic activity of theglycosidase composition or a glycosidase composition, wherein theglycosidase activity represent at least 50% of the total enzymaticactivity of the glycosidase composition.

Many times if would be preferred that such a substantial pureglycosidase composition is a glycosidase composition, wherein theglycosidase activity represent at least 90% of the total enzymaticactivity of the glycosidase composition.

An effective amount of a glycosidase may be one specific type of aglycosidase (e.g. PNGase-F) or be a mixture of herein relevantglycosidase enzymes (e.g. two different N-linked glycosidases or oneN-linked and one O-linked glycosidase).

When there herein is said that it may be preferred that the glycosidaseadded in step (i) of the method of the first aspect is a N-linkedglycosidase it of course means that there must be added an effectiveamount of a N-linked glycosidase in step (i) and this N-linkedglycosidase gives—as a result of its presence—improved gel firmness tothe dairy milk product.

However, when said that N-linked glycosidase is preferred it does ofcourse not mean that there must not be added any O-linked glycosidase instep (i).

The same applies when herein is said that O-linked glycosidase ispreferred—i.e. here there must be added O-linked glycosidase in step(i)—but there may also be added N-linked glycosidase.

To the contrary and as evident to the skilled person—when there hereinis said that the glycosidase is only an O-linked glycosidase then theremust not be added N-linked glycosidase in step (i) of the first aspect.The same applies when herein is said the glycosidase is only an N-linkedglycosidase then there must not be added O-linked glycosidase in step(i) of the first aspect.

As known in the art—a dairy milk product is generally given a heattreatment. As known in the art—heat treatment typically usestemperatures below boiling since at very high temperatures, caseinmicelles will irreversibly aggregate (or “curdle”).

In the present context—the term “pasteurized” in relation to apasteurized dairy animal milk product refers to a standardpasteurization step (i.e. involving a suitable heat treatment of themilk). In the present context—it is evident that the skilled personknows if a specific milk product of interest is a pasteurized dairyanimal milk product or not.

As understood by the skilled person—a standard pasteurization step maybe heat treatment of around 71-72° C. for around 15-20 seconds

As discussed above—to obtain a “yogurt or fermented milk” product it isin particular recalled that there must not be a significant eliminationof milk serum and that there must be a heat treatment at leastequivalent to pasteurization.

A suitable relevant heat treatment for making a fermented milk productsuch as e.g. a yogurt is, for example, a heat treatment of from 85 to98° C. for 15 seconds to 30 minutes.

Depending on the type of heat treatment used—the heat treatment may e.g.be a heat treatment of the milk by using a temperature from 65 to 150°C. for a fraction of a second to 30 minutes.

As discussed above—when the milk product is a fermented milk productsuch as e.g. a yogurt there must be a heat treatment at least equivalentto pasteurization.

The glycosidase may in step (i) be added before or after the heattreatment step—i.e. the heat treatment of the milk by using atemperature from 65 to 150° C. for a fraction of a second to 30 minutes.

In some cases it may be preferred that that glycosidase is added toalready heat treated milk. As discussed above—when the milk product is afermented milk product the milk substrate is inoculated and fermentedwith a relevant lactic acid bacteria (LAB) culture.

When the milk product is a fermented milk product—the glycosidase may beadded before, together or after the inoculation of the milk substratewith the lactic acid bacteria (LAB) culture.

In relation to milk product in general—it may preferred that theglycosidase is added before the pH of the milk substrate gets below pH6.

In relation to a fermented milk product—it is preferred that theglycosidase is added before the relevant lactic acid bacteriafermentation process has ended—i.e. preferably before the pH of the milksubstrate gets below pH 6.

It is herein believed that addition of from 10 activity units per mlmilk to 1000 activity units per ml milk of glycosidase is enough to geta herein relevant effective amount of a glycosidase—i.e. enough to get aherein relevant improved gel firmness.

If relevant for a specific purpose—one may add more glycosidase e.g. upto 20000 activity units per ml milk of glycosidase.

As discussed above—the effective amount/activity of a glycosidase isherein determined according to the art.

Step (ii) of the Method of First Aspect

Step (ii) of the herein described method of the first aspect reads:“(ii): performing adequate step(s) to get a dairy animal milk product,wherein the adequate step(s) is/are performed under conditions, whereinthe effective amount of the glycosidase gives—as a result of itspresence—improved gel firmness to the dairy milk product”

As discussed above—performing adequate step(s) to get a dairy animalmilk product of interest is routine work for the skilled person—e.g. ifthe milk product is e.g. a yogurt, the skilled person of course knowsthe adequate step(s) to get yogurt of interest.

In relation to the conditions, wherein the effective amount of theglycosidase gives improved gel firmness to the dairy milk product—it isevident that these conditions shall be conditions, wherein theglycosidase enzyme as such is active during a sufficient time period.

As shown in the working Examples herein—the present inventors haveidentified that a number of different glycosidase enzymes have asuitable activity during normal suitable conditions (e.g. temperature,pH etc) for making a herein relevant milk product such as a yogurt.

In short and as understood by the skilled person—the preferredconditions to get preferred herein relevant glycosidase activity willdepend on the specific glycosidase enzyme(s) used (e.g. PNGase-F) andthe specific milk product to be made (e.g. a yogurt).

In the present context—examples of suitable reaction conditions for theglycosidase to give the herein relevant improved gel firmness could be:

-   -   Temperature from 10 to 50° C. (such as from 20 to 40° C.);    -   pH from pH 3 to pH 9 (such as from pH 4 to pH 7.5, from 3 to 7        or from 3 to 6.5);    -   a time period from 10 minutes to 120 hours (such as from 1 hour        to 120 hours, or from 0.5 to 5 hours)

In a preferred embodiment, the presence of the glycosidase in step (ii)gives a 1.25 times improved gel firmness to the dairy milk product; morepreferably a 1.50 times improved gel firmness to the dairy milk productand even more preferably the presence of the glycosidase in step (ii)gives a 1.7 times improved gel firmness to the dairy milk product.

As discussed above—the present inventors identified that use of theglycosidase does not negatively affect the viscosity of the final milkproduct.

Accordingly, in a preferred embodiment—step (ii) is performed underconditions, wherein the effective amount of the glycosidase gives—as aresult of its presence—no negative effect on the viscosity to the dairymilk product.

It is routine work for the skilled person to measure viscosity of a milkproduct of interest (e.g. a yogurt).

In working Example 2 herein is provided a suitable standard method formeasurement of viscosity of a milk product of interest—preferably theherein relevant viscosity is measured according to the method of thisExample 2.

The article of A. N. Hassan et al (J. Dairy Sci: 86:1632-1638; 2003)discussed above also describes a suitable standard method formeasurement of viscosity.

See e.g. the materials and method section and FIG. 1 of the article,where shear stress is determined—as can be seen in Example 2 herein, theparameter shear stress is used as a measure of viscosity.

Since it is easy for the skilled person to measure viscosity of a milkproduct of interest—it is of course also easy for the skilled person todetermine the preferred embodiment of step (ii) of the method of thefirst aspect relating to if:

“step (ii) is performed under conditions, wherein the effective amountof the glycosidase gives—as a result of its presence—no negative effecton the viscosity to the dairy milk product”.

In order to determine this requirement—the skilled person shall simplyperform the “adequate step(s) to get a milk product” of step (ii) withand without presence of the effective amount of the glycosidase—and thendetermine the herein relevant viscosity effect of the presence of theadded amount of glycosidase.

As can be seen in the working Examples herein—one may actually get animproved viscosity by using a glycosidase as described herein.

Accordingly, in a preferred embodiment, the presence of the glycosidasein step (ii) gives a 1.10 times increased viscosity to the dairy milkproduct; more preferably a 1.20 times increased viscosity to the dairymilk product.

As understood by the skilled person—the method to measure viscosity ase.g. described in working Example 2 herein and the article of A. N.Hassan et al will (within relatively minor measurement uncertainties)give the same relative results—i.e. independently of the method used onewill get the same result with respect to the relative improvement of theviscosity.

As discussed above—in working Examples herein was demonstrated that bothN-linked and O-linked glycosidase gave no herein unwanted syneresiseffect in relation to making a yogurt.

Accordingly, in a preferred embodiment step (ii) is performed underconditions, wherein the effective amount of the glycosidase gives—as aresult of its presence—no increase in the syneresis effect to the dairymilk product.

This—no increase in the syneresis effect—is particular relevant when thedairy milk product is a fermented milk product such as e.g. a yogurt.

It is routine work for the skilled person to measure syneresis effect ofa milk product of interest (e.g. a yogurt).

In working Example 3 herein is provided a suitable standard method formeasurement of syneresis of a milk product of interest—preferably theherein relevant syneresis is measured in accordance with the method ofthis Example 3.

Essentially, the standard method to measure syneresis effect of Example3 is based on a proper relevant storage of a milk product of interestand measurement of the amount of whey on top of the milk product ofinterest.

Since it is easy for the skilled person to measure syneresis of a milkproduct of interest—it is of course also easy for the skilled person todetermine the preferred embodiment of step (ii) of the method of thefirst aspect relating to if:

“step (ii) is performed under conditions, wherein the effective amountof the glycosidase gives—as a result of its presence—no increase in thesyneresis effect to the dairy milk product”.

In order to determine this requirement—the skilled person shall simplyperform the “adequate step(s) to get a milk product” of step (ii) withand without presence of the effective amount of the glycosidase—and thendetermine the herein relevant syneresis effect of the presence of theadded amount of glycosidase.

Also, the present invention relates to a method for producing a milkproduct, said method comprising:

a) providing a milk substrate;b) treating the milk substrate with an enzyme having N-linkedglycosidase activity; andc) optionally fermenting the milk substrate with a microorganism, suchas a lactic bacterium.

In an interesting embodiment, step b) is performed before or during stepc).

As discussed above, the milk substrate may origin from any animal ornon-animal source.

The milk product is preferably produced substantially without, orcompletely without any addition of a thickener and/or stabilizer, suchas pectin, gelatin, starch, modified starch, carrageenan, alginate, andguar gum.

In an interesting embodiment, the microorganism is a lactic acidbacterium and/or a microorganism which produces a polysaccharide, suchas an exopolysaccharide (EPS).

The microorganism may be a lactic acid bacterium, and preferably belongto a species selected from the group consisting of: Streptococcusthermophilus, Lactobacillus delbrueckii subsp. Bulgaricus, Lactococcuslactis, Lactococcus lactis subsp. cremoris, Leuconostoc mesenteroidessubsp. cremoris, Pseudoleuconostoc mesenteroides subsp. cremoris,Pediococcus pentosaceus, Lactococcus lactis subsp. lactis biovar.diacetylactis, Lactobacillus casei subsp. Casei, Lactobacillus paracaseisubsp. Paracasei, Bifidobacterium bifidum, and Bifidobacterium longum.

Interesting embodiments of any method of the present invention are:

-   -   A method wherein the milk substrate is subjected to        pasteurization before acidification and the enzyme treatment is        performed before pasteurization;    -   A method wherein the milk substrate is subjected to heat        treatment prior to treatment with the enzyme having N-linked        glycosidase activity;    -   A method wherein the milk product is selected from the group        consisting of: a set-type fermented milk product, a drinkable        fermented milk product, and a spoonable fermented milk product;    -   A method wherein the milk product has a milk solid non-fat        content of less than 8%;    -   A method wherein the milk product has a fat content of less than        2%;    -   A method wherein the milk product has a fat content of less than        0.5%;    -   A method wherein the milk product is packaged (ie the method        comprises packaging); and/or    -   A method wherein the glycosidase is selected from the group        consisting of: α-N-acetyl-galactosaminidase; GalNAC);        α-galactosidase; neuraminidase;        Peptide-N(4)-(N-acetyl-beta-glucosaminyl)asparagine amidase;        N-Glycosidase-F; PNGase-F; Endo-β-N-acetylglucosaminidase H;        ENDO-H and any enzyme classified in EC 3.2.1.49, EC 3.2.1.18, EC        3.2.1.22, EC 3.5.1.52, or EC 3.2.1.96.

In the present context, the term “packaging” (a suitable amount of) theproduct in a suitable package relates to the final packaging of theproduct to obtain a product in distributable form so that the productcan be ingested by e.g. a person or a group of persons. A suitablepackage may thus be a bottle, container, package or similar, and asuitable amount may be e.g. 10 ml to 5000 ml, but it is presentlypreferred that the amount in a package is from 50 ml to 1000 ml. Such apackaged product is a part of the present invention.

In a further aspect, the present invention also relates to a milkproduct obtainable by a method of the present invention.

In yet a further aspect, the present invention relates to a milk productwhich is obtainable by a method comprising adding an N-linkedglycosidase and/or a O-linked glycosidase to a milk substrate. Theglycosidase should be added in “an effective amount” to give the desiredgel stiffness.

The milk products of the invention may have been fermented byinoculation with a lactic acid bacteria culture, prior to and/or duringand/or after the treatment with the glycosidase.

The milk product may be packaged, e.g. in a sealed container having avolume in the range of 10 ml to 5000 ml, such as in a container having avolume of 25 ml to 1500 ml or a volume of 50 ml to 1000 ml.

In a further aspect, the present invention relates to the use of anN-linked glucosidase in a method for preparation of a milk product, suchas a fermented milk product (such as yoghurt) or cheese (such as freshcheese, fromage frais, quark, etc).

Also, the present invention relates to the use of an N-linkedglucosidase in a method for improving the texture (such as gel firmnessor stiffness) of a milk product, such as a fermented milk product (suchas yoghurt) or cheese (such as fresh cheese, fromage frais, quark, etc).

In a presently preferred embodiment, the invention relates to the use ofan enzyme having N-linked and/or O-linked glycosidase activity forimproving the mouth feel of a milk product, such as yoghurt.

The use may comprise that the milk product has been produced using alactic acid bacterium which produces a polysaccharide, such as anexopolysaccharide (EPS).

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising”, “having”, “including” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

EXAMPLES Example 1 Glycosidases

The examples of glycosidases used in the method of as described hereinis an asparagine amidase, an acetylglucosaminidase or agalactosaminidase.

The N-Glycosidase-F, also known as PNGase-F, used in the process, is anasparagine amidase (EC 3.5.1.52) which may be derived fromFlavobacterium mesingosepticum. It catalyses the complete and intactcleavage of N-linked oligosaccaharides from glycoproteins. It may beobtained as a commercial product from New England Biolabs Inc. under thename PNGase-F or produced recombinantly in a strain like Escherichiacoli as we have done using the plasmid and method described in Loo et at(Protein Expression and Purification 24, 90-98, 2002).

Endo-β-N-acetylglucosaminidase H (EC 3.2.1.96), also known as ENDO-H,may be derived from e.g. Streptomyces plicatus. ENDO-H catalyses thehydrolysis of the glycosidic bond between the two N-acetylglycosaminesof N-linked glycosylations. It may be obtained as a commercial productfrom New England Biolabs Inc. under the name ENDO-H.

α-N-acetyl-galactosaminidase (EC 3.2.1.49) is a highly specificexoglycosidase that catalyzes the hydrolysis of α-linkedD-N-acetyl-galactosamine residues from threonines or serines. It may beobtained as a commercial product from New England Biolabs Inc. under thename the name α-N-acetyl-galactosaminidase.

For the in-house produced PNGase-F we have confirmed a high grade (>95%)of purity using Sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE) followed by coomassie brilliant blue (CBB)staining. To further confirm the purity the pooled enzyme was separatedby SDS-PAGE and stained using silver. Appearing bands were next analyzedby mass-spec analysis which confirmed the presence of PNGase-F and onlyPNGase-F.

Potential a-specific proteolytic activity of the in-house purifiedPNGase-F was evaluated in a proteolytic assay using bovine-kappa-casein(Sigma-Aldrich) as a substrate. In this assay we tested the proteolyticactivity at various pH values resembling the natural pH drop occurringin yogurt fermentation. To challenge the system we utilized a 20 foldhigher enzyme concentration than in the process of the invention andincubated four hours at each pH-value. Under these conditions we did notfind any evidence of a-specific proteolytic activity.

Conclusions:

Based on the results discussed above—it was clear that the so-calledin-house produced PNGase-F was free of contaminants.

The other commercially available glycosidases discussed above were alsofree of contaminants as described by the supplier.

Example 2 Method for Measuring Gel Firmness and Viscosity

Gel firmness was measured by the use of an Anton Paar rheometer with anautomatic sample changer (Physica DSR Rheometer+ASC). The measuring bobwas placed in the measuring cup containing 20 ml sample, which had beenstirred by the hand and heated to 13° C. After the bob had been placedin the sample a wait time was applied. Next, gel firmness was measuredby oscillation. Here the strain was kept constant at 0.3% and thefrequency was increased from 0.5 Hz to 30 Hz. From the measurement theelastic modulus (G′) and the viscous modulus (G″) could be calculated,and from these the complex modulus (G*) was obtained:

G*=√{square root over (G ^(′2) +G ^(″2))}

G* at 1 Hz was then used as a measure of the gel firmness and used forcomparison of the different samples.

Using the same equipment (Anton Paar rheometer) the viscosity wasmeasured by increasing the shear rate from 0.2707 l/s to 300 l/s withmeasuring points (shear stress) every 10 s. The shear rate was thendecreased from 275 l/s to 0.2707 l/s with measuring points every 10 s.Shear stress at 300 l/s was then used as a measure of the viscosity ofthe product.

Conclusions:

Based on above standard methods for measuring gel firmness andviscosity—it is routine work for the skilled person to determine ifthere has been an improvement of the gel firmness and/or viscosity to adairy milk product of interests by addition of a glycosidase accordingthe method for making a dairy animal milk product as described herein.

Example 3 Methods for Measuring Syneresis

Two different methods for measuring the amount of syneresis have beenused in the invention:

Method 1:

50 ml yogurt (produced like in example 4) is put into an Eppendorf tubeand placed in cold storage (5° C.) for 14 days, after which the amountof whey on top of the yogurt is measured with a ruler (in mm).

As evident to the skilled person—one may also measure the syneresiseffect of another milk product than yogurt by storage of a milk productof interest and measure the amount of whey on top of the milk product ofinterest.

Method 2:

Skim milk was fortified with 2% skim milk powder (SMP, producer?) andplaced in the refrigerator overnight. The batch was heat treated for 20minutes at 90° C. 75 ml milk solution was put into a 100 ml volumetricflask together with 0.02% YoFlex® Advance 2.0 yogurt culture (may beobtained from Chr. Hansen A/S, Denmark) and enzyme (either PNGase orGalNAC). The total weight of the milk, culture and enzyme was noted. Thesolution was heated to 43° C. and fermented to pH 4.55 after which theflasks were put in cold storage for 7 days. After 7 days the whey on topof the yogurt was poured of and weighed. The amount of syneresis canhereafter be calculated as a percentage.

As evident to the skilled person—one may also measure the syneresiseffect of another milk product than yogurt by e.g. fermenting withanother not yogurt culture of interest, storage and measure the amountof whey on top of the milk product of interest.

Conclusions:

Based on above standard methods for measuring syneresis effect—it isroutine work for the skilled person to determine if there has been asignificant syneresis effect to a dairy milk product of interests byaddition of a glycosidase according the method for making a dairy animalmilk product as described herein.

Example 4 Effect of PNGaseF on Gel Firmness (2 l Scale)

2 liters of skim milk (0.1% fat, Arla Express, Slagelse) was fortifiedwith 1.6% skim milk powder (SMP, producer?) and placed in therefrigerator overnight. The solution was heat treated for 20 minutes at90° C., cooled down to 43° C., inoculated with 0.02% YoFlex® Advance 2.0(obtained from Chr. Hansen A/S, Denmark) and either 10 ml PNGase-F or inthe reference sample 10 ml 20 mM Na-phosphate buffer pH 7.5 containing50 mM NaCl and fermented to pH 4.55. When pH 4.55 was reached, theyogurt was stirred and passed through a post treatment unit (PTU), whichsubjects the yogurt to a back pressure of 2 bars at 25° C. The finalproduct was subjected to rheometer analysis on day 5.

TABLE 1 Experimental results of a 2 I scale experiment of thepreparation of a low fat yogurt in the absence and presence of in-housePNGase-F Complex Shear stress at Syneresis Modulus (Pa) (300 1/s) (mm)Advance 2.0 29.2 ± 0.86 63.7 ± 0.61 8 Advance 2.0 + PNGase-F 51.0 ± 0.5770.7 ± 0.2  4 (250 U/ml milk)

For the N-glycosidases one activity unit is defined as the amount ofenzyme required to remove >95% of the carbohydrate from 10 μg ofdenatured RNase-B in 1 hour at 37° C. in a total reaction volume of 10μl

The numbers in table 1 show that PNGase-F increases the gel firmness ofa low fat yogurt. Under these conditions with 75% compared to thereference. The viscosity obtained in the presence of PNGase-F iscomparable or slightly better than the reference sample. In data notshown here we found that the increase in gel firmness in response toPNGase-F treatment was dose dependent. We also evaluated syneresis usingmethod 1 from Example 3 and found that the syneresis was reduced inresponse to PNGase-F treatment.

Conclusions:

The results above demonstrated that PNGase-F increases the gel firmnessof a low fat yogurt. Under the conditions of this experiment with 75%(i.e. 1.75 times) compared to the reference. Further was found that thesyneresis was reduced in response to PNGase-F treatment.

The viscosity obtained in the presence of PNGase-F is comparable orslightly better than the reference sample.

Example 5 Testing of Different Glycosidases in Low Fat Yogurt

Yogurts were made directly in 20 ml rheometer cups. Hereby a set-yogurtwas obtained. Skim milk (0.1% fat, Arla Express, Slagelse) was fortifiedwith 2% skim milk powder (SMP) and placed in the refrigerator overnight.The solution was heat treated for 20 minutes at 90° C., cooled down to43° C., inoculated with 0.02% YoFlex®Advance 2.0 and differentdeglycosidases and fermented (Table 1)

The samples were then placed in the refrigerator and the rheology wasmeasured at day 3 according to example 2.

TABLE 2 Experimental result of a 20 ml cup lab scale experiment of thepreparation of a low fat yogurt in the presence and absence ofcommercially obtained deglycosidases. All tested enzymes in this tablewere obtained from New England Biolabs Inc. Concentration Gel firmness(U/ml milk) (Pa) Advance 2.0 + PNGase-F 250 608 ± 32.5 Advance 2.0 +Endo-H 250 564 ± —  Advance 2.0 + α-N-Acetyl 50 482 ± 19.8galactosaminidase Advance 2.0 0 391 ± 7.5 

For N-glycosidases PNGase-F and Endo-H one unit is defined as inTable 1. For α-N-Acetyl-galactosaminidase, an O-glucosidase, one unit isdefined as the amount of enzyme required to cleave >95% of the terminalα-D-N-acetyl-galactosamine from 1 nmol(GalNAca1-3)(Fucα1-2)Galα1-4Glc-7-amino-4-methyl-coumarin (AMC), in 1hour at 37° C. in a total reaction volume of 10 μl.

For all tested glycosidases, N-glucosidases as well as the O-glucosidaseGalNAC, the gel firmness was found to be improved markedly. At thetested concentrations PNGase-F obtained commercially was found to havethe most potent effect on the gel firmness of the low fat yogurt.

Conclusions:

The results above demonstrated that for all tested glycosidases,N-glucosidases as well as the O-glucosidase GalNAC, the gel firmness wasfound to be improved markedly.

Example 6 Syneresis Experiment Using a Glycosidase Working on O-LinkedGlycosylations

In U.S. Pat. No. 7,560,127 it was shown that the α-glucosidase GalNACcould lead to cheese curd/clotting formation. We here analyzed whateffect GalNAC had on syneresis formation in low fat yogurt. Theexperiment was conducted as described in Example 3 Method 2. Yogurtswere made directly in 100 ml rheometer cups. Hereby a set-yogurt wasobtained. Skim milk (0.1% fat, Arla Express, Slagelse) was fortifiedwith 2% skim milk powder (SMP, Aria Express, Slagelse) and placed in therefrigerator overnight. The solution was heat treated for 20 minutes at90° C., cooled down to 43° C., inoculated with 0.02% YoFlex® Advance 2.0and GalNAC at a concentration of 100 U/ml of milk. For the referencesample without enzyme the percentage of whey after 21 days was 0.3±0.04%whereas the whey fraction for the enzyme treated sample was 0.2±0.08%.Surprisingly, it was therefore found that treatment with GalNAC did notincrease the syneresis when compared to a reference sample fermentedwith the same culture. This result was unexpected since U.S. Pat. No.7,560,127 would indicate that removal of O-linked glycans leads to curdformation and thus separation of the whey.

Conclusions:

The results above demonstrated that the tested O-linked glycosidase didnot increase the syneresis during the production of the yogurt.

Example 7 LAB and Chemically Acidified Milk

In the current example we wanted to assess whether or not the effect ofPNGase-F was dependent on the mechanism of acidification. Experimentallythis was addressed by comparing the effect of PNGase-F on a fermentedyogurt and a yogurt acidified chemically with Glucono-δ-lactone (GDL).

A milk consisting of 9.5% dry matter, was inoculated with eitherYoFlex®Advance 2.0 or Glucono-δ-lactone (GDL) and PNGase-F as indicatedin Table 3, heated to 43° C. and fermented to pH 4.55. After samples hadbeen stored for 3 days at 5° C., the samples were stirred with a stirrerand poured into the rheometer cups and the rheology was measuredaccording to example 2 and 3.

The results presented in table 3 confirmed that the effect of PNGase-Fmay be described as a physical phenomenon. The chemically acidifiedyogurt generates a much firmer gel than the Advance 2.0 culture.However, for both methods of acidification it is evident that theaddition of PNGase-F improves the gel firmness.

TABLE 3 Experimental result of a 200 ml lab scale experiment of thepreparation of a low fat yogurt in the presence of PNGase-F using eitherAdvance 2.0 or GDL for acidification Concentration Gel Firmness (U/mlmilk) (Pa) Advance 2.0 + PNGase-F 250  35.3 ± 7.8 Advance 2.0 0  24.7 ±0.9 GDL + PNGase-F 250 148.0 ± 7.0 GDL 0 121.5 ± 6.4

Conclusions:

The results above demonstrated for both methods of acidification it isevident that the addition of PNGase-F improved the gel firmness.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

REFERENCES

-   1. US2005/0095316A1 (Danone)-   2. US2005/0095317A1 (Danone)-   3. WO2007/095958A1 (Chr. Hansen A/S)-   4. A. N. Hassan et al (J. Dairy Sci: 86:1632-1638; 2003)-   5. U.S. Pat. No. 7,560,127B2 (DSM)-   6. E. Cases et al (Journal of Food Science; Vol. 68, Nr. 8, 2003,    Pages 2406-2410)-   7. EP1489135A1-   8. R. Scott, (1986), Cheesemaking process, second ed., Elsevier    Applied Science Publishers, London and New York.-   9. G. Bylund, (1995), Dairy processing handbook, Tetra Pak    Processing Systems, Lund, Sweden-   10. F. Kosikowski, (1982), Cheese and fermented milk foods, second    ed., Kosikowski & Associates, New York

All references cited in this patent document are hereby incorporatedherein in their entirety by reference.

1-36. (canceled)
 37. A method for producing a yogurt product comprising:(a) treating a milk substrate with an enzyme having O-linked glycosidaseactivity and (b) fermenting the milk substrate, to produce a yogurtproduct that has increased gel firmness relative to a yogurt productproduced with a comparable method but without the enzyme having O-linkedglycosidase activity.
 38. The method of claim 37, wherein step (a) isperformed before or during step (b).
 39. The method of claim 37, whereinthe enzyme having O-linked glycosidase activity is selected from thegroup consisting of: α-N-acetyl-galactosaminidase (EC number: 3.2.1.49);α-galactosidase (EC number: 3.2.1.22); neuraminidase (EC number:3.2.1.18); and combinations thereof.
 40. The method of claim 37, whereinthe milk substrate is selected from the group consisting of milk fromanimals and milk of plant origin.
 41. The method of claim 40, whereinthe milk from animals is from cows, sheep, ewes, goats, buffaloes, orcamels.
 42. The method of claim 40, wherein the milk of plant origin issoy milk, oak milk, rice milk, or almond milk.
 43. The method of claim37, wherein the fermenting is with lactic acid bacteria.
 44. The methodof claim 37, wherein the fermenting is with a microorganism that belongsto a species selected from the group consisting of: Streptococcusthermophilus, Lactobacillus delbrueckii subsp. bulgaricus, Lactococcuslactis, Lactococcus lactis subsp. cremoris, Leuconostoc mesenteroidessubsp. cremoris, Pseudoleuconostoc mesenteroides subsp. cremoris,Pediococcus pentosaceus, Lactococcus lactis subsp. lactis biovar.diacetylactis, Lactobacillus casei subsp. casei, Lactobacillus paracaseisubsp. paracasei, Bifidobacterium bifidum, and Bifidobacterium longum.45. The method of claim 37, further comprising adding lactic acidbacteria to the milk substrate.
 46. The method of claim 45, wherein 10⁵to 10¹³ CFU/g of the lactic acid bacteria are added to the milksubstrate.
 47. A yogurt product obtained by the method of claim 37.